Adenylate cyclase (AC) toxin is a novel virulence factor for several species of the genus Bordetella, including B. pertussis (whooping cough, pertussis in humans), B. parapertussis (different strains causing diseases in animals and humans) and B. bronchiseptica (kennel cough in dogs, and respiratory diseases in most mammals). Its major function has been considered to be the ability to translocate its AC domain across the cytoplasmic membrane of target cells and produce supraphysiologic quantities of cAMP. We have now demonstrated, however, that the ability of AC toxin to form transmembrane pores is also relevant to cytotoxicity for macrophages. Furthermore, simply binding of the toxin to CR3, a leukocyte integrin that can act as a receptor for AC toxin, can activate human neutrophils (PMN), independent of its cAMP production and pore formation. In this renewal proposal, we will develop these recent discoveries and focus on the mechanisms and target tissues through which AC toxin is indispensable to B. pertussis in the establishment of infection in the respiratory tract. We will use in vitro approaches with either purified toxin or bordetella organisms (both B. pertussis and B. bronchiseptica) to learn more about toxin composition, packaging, secretion and delivery (SA I), the effects of the toxin on the two prime candidate target tissues, leukocytes (SA II) and the respiratory epithelium (SA III), through its three functions (occupancy of integrin receptor, cAMP production and pore formation). Finally, in order to begin to relate the actions of AC toxin to those of other important virulence factors, we will evaluate other toxins (pertussis toxin, tracheal cytotoxin) and adhesins (filamentous hemagglutinin, pertactin) from B. pertussis, both individually and in conjunction with AC toxin, to understand better the possible interactions (enhancement, synergy, antagonism or no effect) of their effects in leukocytes and the respiratory epithelium (SA IV). The objective is to apply the information on AC toxin and on other bordetellae virulence factors, to a more focused study of how AC toxin contributes to infections and diseases caused by this genus.